The invention relates to an optical arrangement in the beam path of a confocal fluorescence microscope, having at least one laser light source, a device arranged in the illumination/detection beam path to separate the exciting light reflected at the object from the fluorescent light radiated by the object, an objective arranged between the device and the object, and a detector arranged downstream of the device in the detection beam path.
In practice, it has already been known for years to use a dichroic beam splitter to separate the exciting light reflected at the object from the fluorescent light radiated by the object. In the simultaneous multicolor application, a number of dichroic beam splitters are correspondingly used. The fluorescent light freed of the reflected exciting light is detected by means of a special detectorxe2x80x94after being separated by means of beam splitters. The beam splitters used in practice are generally expensive. Furthermore, these beam splitters are only little suited to quantitative comparative measurements at high precision and with high measurement dynamics, especially since these beam splitters are, inter alia, also temperature-dependent. Furthermore, dichroic beam splitters have transmission losses of about 10% for the detection.
If one were to use, for example, a beam splitter that was independent of wavelengths, this would doubtless reduce the costs. However, the disadvantage in this case would be that it would be necessary to filter out the scattered light from the exciting light before detection, for example by using a blocking filter. This is again complicated from a constructional point of view and once again gives rise to costs. In addition, the result is that the fluorescence yield is reduced. However, simultaneous multicolor applications are then not possible.
If a simple beam splitter is used, in addition reflected scattered light from the exciting light gets back into the laser light source and interferes with the stimulated emission taking place there, which in turn makes itself noticeable through undesired intensity fluctuations in the laser light.
For the highest resolution applications the same numerical aperture is used for the illumination and for scanning. This leads to an illumination focus which is very small in the lateral direction, so that relatively long recording times are necessary. For applications in which the resolution plays a subordinate role, this procedure is disadvantageous because of the longer recording times.
With regard to confocal fluorescence microscopy, reference is made, merely by way of example, to Engelhardt and Knebel in xe2x80x9cPhysik in unserer Zeitxe2x80x9d [Physics in our time], Vol. 24, 1993, number 2 xe2x80x9cConfocal laser scanning microscopyxe2x80x9d, and D. K. Hamilton and T. Wilson in Appl. Phys. B 27, 1982, 211-213 xe2x80x9cThree-dimensional Surface Measurement Using the Confocal Scanning Microscopexe2x80x9d.
The present invention is, then, based on the object of configuring and developing an optical arrangement in the beam path of a confocal fluorescence microscope in such a way that it is possible to implement an increase in the fluorescence yield with a simple design by comparison with the generic arrangement with a conventional dichroic beam splitter.
The optical arrangement according to the invention in the beam path of a confocal fluorescence microscope achieves the above object by means of the features of patent claim 1. According to this claim, the optical arrangement mentioned at the beginning is characterized in that the device comprises a mirror, and in that the mirror is dimensioned and arranged in the illumination/detection beam path in such a way that, for dark-field illumination of the object, it reflects the non-widened exciting beam coming from the laser light source into the objective and permits the fluorescent light coming from the object to pass in the direction of the detector with full numerical aperture, reduced by the effective cross section of the mirror in the detection beam path.
According to the invention, it has firstly been found that the device comprises a mirror, and in that the mirror is dimensioned and arranged in the illumination/detection beam path in such a way that, for dark-field illumination of the object, it reflects the non-widened exciting beam coming from the laser light source into the objective and permits the fluorescent light coming from the object to pass in the direction of the detector with full numerical aperture, reduced by the effective cross section of the mirror in the detection beam path.
According to the invention, it has further been found that the device for separating the exciting light reflected at the object from the fluorescent light radiated by the object can bexe2x80x94instead of a conventional dichroic beam splitterxe2x80x94a mirror which is arranged in the illumination/detection beam path. In this case, this mirror is to be dimensioned in such a wayxe2x80x94sufficiently smallxe2x80x94that, for dark-field illumination of the object, it reflects the non-widened exciting beam coming from the laser light source into the object and permits the fluorescent light coming from the object to pass in the direction of the detector with full numerical aperture, the fluorescent light being reduced by the effective cross section of the mirror in the detection beam path. The main reflection of the exciting light reflected by the object is advantageously reflected out of the detection beam path at the mirror.
Because of the sufficiently small configuration of the mirror or of the reflective area, only the non-widened exciting beam is reflected into the objective. By illuminating with a xe2x80x9csmallxe2x80x9d numerical aperture of this type (for example 10% of the otherwise usual aperture), xe2x80x9cdark-field illuminationxe2x80x9d is achieved which exhibits an elongated focal range with a well-defined focal diameter along the optical axis. According to the invention, an adequate illumination tolerance in the object position along the optical axis is achieved, which has a particularly beneficial effect on specific applications in confocal fluorescence microscopy. Furthermore, illumination with high intra-scene dynamics is ensured. The advantage of such high intra-scene dynamics is that, for example, two immediately adjacent objects with different absolute fluorescent intensities, for example object A at 100% and object B at 0.05%, can be measured separately from each other in the same confocal plane without xe2x80x9cover-illuminatingxe2x80x9d the lighter object.
In a particularly advantageous way, the mirror is dimensioned and configured to be small in such a way that, for the fluorescent light to be detected, it causes a loss in the detection beam path of about 1%. This permits efficient detection with a particularly high dynamic range.
Within the context of a particularly simple embodiment, the mirror used here could be designed as an independent component, the mirror in turn being carried by a holder. Within the context of such a configuration, the use of a conventional beam splitter is completely dispensed with, since here only the small mirror is specifically arranged at an appropriate point in the beam path. Within the context of a further alternative, the mirror could be designed as a preferably integrally silvered area of anxe2x80x94otherwise conventionalxe2x80x94beam splitter, the mirror or the silvered area being arranged or formed at least largely at the center of the beam splitter. This smallxe2x80x94integralxe2x80x94mirror could be approximately circular or elliptical or oval. Ultimately, what is concerned here is a silvered area at the center of a beam splitter which, just like an isolated mirror, reflects the non-widened exciting beam into the objective. Here, too, illumination takes place with an extremely small numerical aperture, to the benefit of the dynamics.
The non-reflective area of the beam splitter could exhibit approximately 10% reflection and 90% transmission in the direction of the detector. In concrete terms, this could be an anti-reflection (AR) coating on that side of the beam splitter facing the detector. This beam splitter permits the fluorescent light to pass in the direction of the detector in accordance with the conventionalxe2x80x94infinitexe2x80x94beam path at full numerical aperturexe2x80x94reduced by the fluorescent light incident on the silvered area. The silvered area produces an extremely low loss for the fluorescent light to be detected, specifically depending on the size of the mirror. Here, it is possible to realize losses of only 1%, which leads to the high dynamics already previously mentioned.
Moreover, the non-reflective area of the beam splitter could be configured and, if appropriate, coated in such a way that the optical properties of the non-reflective area of the beam splitter are at least largely independent of temperature. This is to the benefit of the reproducibility of the measurement.
Now, it is not only fluorescent light which passes from the object into the detection beam path, but also back-reflection of the exciting light. Such back-reflection is undesired. In order to suppress the back-reflection of the exciting light at the object back into the laser light source, the object could be arranged crookedly. This means that the object itself is not arranged orthogonal to the optical axis, so that the result for the back-reflected exciting light is a separate beam path which is offset at least slightlyxe2x80x94physicallyxe2x80x94with respect to the illumination beam path. It is readily possible to arrange conventional light traps in this special beam path, specifically in order to mask out the back-reflected exciting light effectively.
In the event of a desired object displacement, for example in order to find a further object, it is of particular advantage if the object can be moved in its plane in such a way that the illuminated object area is always at the same distance from the objective. As a result, the light reflected and/or scattered at the object according to the above embodiments is not reflected back into the laser light source but into a light trap via the special beam path.
As an alternative to the precaution of a light trap, the mirrorxe2x80x94within the context of a configuration as an isolated componentxe2x80x94could have an absorbing area to absorb the exciting light scattered and/or reflected by the object. In this case, the precaution of special light traps is no longer necessary, which results in a reduction in the physical size of the arrangement. Thus, the mirrorxe2x80x94within the context of a circular or elliptical or oval configurationxe2x80x94could have a reflective area and an absorbing area.
It is likewise possible for the beam splitter, apart from the silvered area, to have an absorbing area to absorb the exciting light scattered and/or reflected by the object. Within the context of such a configuration, the precaution of a special light trap can also be dispensed with, so that this configuration also involves the advantage of being able to construct the arrangement with the smallest space requirement.
Finally, it is of quite particular advantage if, by means of a suitable ratio between beam divergence and wavelength of the laser light sources used, an at least approximately identicalxe2x80x94axialxe2x80x94focal position is possible. As a result, on the one hand the necessary illumination tolerance in the object position along the optical axis is provided and, on the other hand, illumination with high intra-scene dynamics is ensured. For this purpose, reference is made to the above embodiments to avoid repetition.
There are then various possibilities of configuring and developing the teaching of the present invention in an advantageous way. For this purpose, reference should be made to the following explanation of exemplary embodiments of the invention, using the drawing. In connection with the explanation of the preferred exemplary embodiments of the invention using the drawing, configurations and developments of the teaching which are generally preferred will also be explained. In the drawing:
There are then various possibilities of configuring and developing the teaching of the present invention in an advantageous way. For this purpose, reference should be made on the one hand to the claims subordinate to patent claim 1 and on the other hand to the following explanation of exemplary embodiments of the invention, using the drawing. In connection with the explanation of the preferred exemplary embodiments of the invention using the drawing, configurations and developments of the teaching which are generally preferred will also be explained. In the drawing